At this Location

1a. Objectives (from AD-416)
We hypothesize that walnuts will reverse age-related disturbances in behavior, neuronal functioning, and microglial activation and will test this hypothesis with the following aims: 1) Determine the effects of dietary supplementation with walnuts or walnut components on motor and cognitive function. 2) Determine the efficacy of walnuts or walnut components on reversing the age-associated increase in neuronal vulnerability. 3) Investigate the anti-inflammatory actions of walnuts and walnut components on microglial activation. These studies will serve to characterize the effects of dietary walnut supplementation in aging, focusing on cognitive, behavioral, neuronal, and microglial effects of walnuts. In addition, these studies will examine the neuroprotective effects of individual walnut components in order to determine the efficacy of the whole food versus individual components of walnuts.

1b. Approach (from AD-416)
1) Determine the effects of dietary supplementation with walnuts or walnut components on motor and cognitive function. Walnuts will be supplemented in the diet at 6%, walnut components will be given as a portion of the 6% concentration that they comprise in the whole food. The effects of these diets on age-sensitive motor (probes sensitive to balance, strength, and coordination) and cognitive behaviors (short- and long-term memory) will then be examined in Fischer 344 (F344) rats after 8 weeks of supplementation.
2) Determine the efficacy of walnuts or walnut components on reversing the age-associated increase in neuronal vulnerability. The effects of dietary supplementation with walnuts and walnut components on hippocampal neurogenesis (proliferation and survival of newly born neurons) will be assessed in subjects utilized for Aim 1. Additionally, the effects of walnuts on neuronal stress and protective signaling will be accomplished through the use of a hippocampal cell line. Hippocampal cells will be used to determine if walnuts and walnut components alter neuronal stress and protective signaling pathways which have been reported to be altered in the aging brain.
3) Investigate the anti-inflammatory actions of walnuts and walnut components on microglial activation. Prolonged microglial activation is a hallmark of neurodegenerative disorders as well as normal brain aging. The effects of dietary walnut supplementation on age-associated microglial activation will be quantified in tissues harvested from subjects in Aim 1. Further studies on the molecular mechanisms of the anti-inflammatory properties of walnut oil and walnut components will be undertaken in the BV2 microglial cell line.

3. Progress Report
We had shown previously under this agreement that walnut supplementation was effective in reversing the cognitive deficits in aged rats as compared to their non-supplemented counterparts. This year we assessed the effects of walnuts on a particular type of brain cell called a microglial cell that contributes to the “clean-up” of damaged cells in the brain. However, in aging and neurodegenerative diseases, these cells can become overactivated and begin to clean up healthy cells. We used BV-2 mouse microglial cells and showed that walnuts deactivate the cells and prevent cell death. We showed that these treatments are also effective in shutting down stress signals in hippocampal cells which are important in memory function reduced inflammatory markers and enhanced cellular calcium function in these cell models. To determine the components of walnuts that may be having beneficial effects in cells, we exposed BV-2 cells to inflammatory stressors to determine the protective roles of polyunsaturated fatty acids (PUFAs), specifically the omega-6 fatty acid linoleic acid (LA) and the omega-3 fatty acid, alpha-linolenic acid (ALA). PUFAs are essential components of neuronal cell membranes, maintaining membrane function critical for neurotransmitter communication within neural networks. Importantly, the extracts appeared to cause internalization of a particular receptor called a Toll-like receptor (TLR). When these receptors were taken inside the cell by the walnut extracts, the access of inflammatory agents such as lipopolysaccharide to the receptor was blocked. This receptor is necessary for the inflammatory process to continue. The effects of walnut extract on BV-2 cells are novel and demonstrate that fruits and nuts can induce dramatic alterations in the stress responses of cells involved in age-related cognitive decline.